Approximately 1 to 15% of patients treated for a cancer by radiotherapy show a tissular reaction (such as dermatitis or proctitis) which can affect the successful provision of the treatment, to the extent that it may lead the physician to decide to stop the radiotherapy treatment before the end of the envisaged protocol. In addition, said tissular reaction is an indicator of a particularly high sensitivity of the patient to ionizing radiation. Moreover, radiotherapy treatment, even if broken off when the first visible tissular signs appear, can increase post-treatment morbidity or even mortality of patients, not only because it has not been possible to totally eradicate the cancer which was intended to be treated due to the premature stopping of the treatment, but also due to collateral damage to healthy tissues induced by the radiation itself.
It is also known that the question of the sensitivity of tissues to ionizing radiation is inseparable from that of the mechanisms of DNA damage repair. Indeed, at the cellular level, ionizing radiation can break some types of chemical bonds by generating free radicals (in particular by peroxidation) and other reactive species that are responsible for damage to DNA. Damage to DNA by endogenous or exogenous attack (such as by ionizing radiation and free radicals), can lead to various types of DNA damage as a function, in particular, of the energy deposited: damage to bases, single-strand breaks and double-strand breaks (DSB). Unrepaired DSB are associated with cell death, toxicity and, more specifically, radiosensitivity. Poorly repaired DSB are associated with genomic instability, mutagenic phenomena and a predisposition to cancer. The body has a specific repair system for each type of DNA damage. Mammals possess two main methods of DSB repair: repair by suture (ligation of the strands) and repair by recombination (insertion of a homologous or non-homologous strand).
It is known that the sensitivity of tissues to ionizing radiation is highly variable from one organ to another and from one individual to another; the concept of an “intrinsic radiosensitivity” was described by Fertil and Malaise in 1981. Moreover, various studies on the therapeutic effects and the side effects of radiotherapy have shown that some individuals enjoy a particularly high radioresistance, whereas other individuals, by contrast, exhibit a radiosensitivity that can lead to clinically recognized side effects, but without leading to a consequent lethal effect. Even excluding certain rare cases of extreme radiosensitivity, the genetic origin of which appears proven, it is thought that radiosensitivity generally stems from a genetic predisposition and is therefore specific to an individual. It would therefore be desirable to have a predictive testing method in order to be able to determine the maximum cumulative dose that a given patient can receive without risk. This question primarily arises in radiotherapy in the context of high ionizing doses. However, the question is also likely to be asked for any other exposure to strongly ionizing doses, equivalent to those used in radiotherapy.
It is known that two proteins from the family of kinases, commonly known as ATM and ATR, are involved in the detection, repair and signaling of DSB; their action requires at least the presence of a protein known by the designation BRCA1 and of a cascade of ordered phosphorylations of various ATM substrates (see the article by N. Foray et al., “A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA 1 protein”, published in The EMBO Journal vol. 22(11), p. 2860-2871 (2003)). A trial has been undertaken to use the ATM enzyme in an explanatory model of cellular radiosensitivity (see Joubert et al., “DNA double-strand break repair defects in syndromes associated with acute radiation response; At least two different assays to predict intrinsic radiosensitivity?”, published in Int. J. Radiat. Biol., vol. 84(2), p. 107-125 (2008)), and this has enabled three types of radiosensitivity to be identified: radioresistant cells (termed Group I radiosensitivity), moderately radiosensitive cells (termed Group II radiosensitivity), and extremely radiosensitive cells (termed Group III radiosensitivity). However, no predictive model has been proposed on this basis. In particular no quantitative relationship has been established between the clinical data (tissular severity grade 0 to 5) and the radiobiological data. Similarly, the presentation of N. Foray, “Les réparatoses: nouveaux concepts sur la prédiction de la radiosensibilité”, delivered during “Rencontres Nucléaire & Santé” on 25 Jan. 2008 (XP55131242) discusses the role of various markers pH2AX and MRE11 and their change over time in order to describe the number of radiation-induced double-strand breaks. This presentation does not mention the grades of tissular severity which quantify and list the level of radiosensitivity observed at the clinical level.
Many documents describe the conditions under which ATM can contribute to the detection and repair of DNA damage. Patent application WO 2004/013634 (KUDOS Pharmaceuticals Ltd) describes the identification of a component of the signaling pathway for ATM-dependent DNA damage, which interacts with other response factors to DNA damage, including the complex MRE11/Rad51/NBS1. Patent application US 2007/0072210 (Ouchi and Aglipay) proposes a method for screening potential therapeutic agents which promote a response to DNA damage, in which a protein known as BAAT1 (which is associated with a predisposition to breast cancer linked to the gene BRCA1) is mixed with an ATM protein and the candidate compound; if the phosphorylation of the ATM is increased with respect to a control mixture which does not contain the candidate compound, the latter is identified as a potential active ingredient promoting DNA repair. Patent application EP 2 466 310 A1 (Helmholtz Zentrum München) describes repair of double-strand breaks in DNA in the presence of the phosphorylated form of the histone H2AX (known as gamma-H2AX or g-H2AX). Application WO 00/47760 and patent U.S. Pat. No. 7,279,290 (St. Jude's Children's Research Hospital) describe the role of the kinase functional group of ATM in DNA repair.
These documents thus describe repair pathways but do not present any correlation to establish a link with clinical data.
Patent EP 1 616 011 B1 (International Centre for Genetic Engineering and Biotechnology) discloses a method for diagnosing a genetic defect in DNA repair, based on three steps: culturing of cells isolated from a sample to be tested, incubating said cells with a chimeric polypeptide, and characterizing the cellular response. Said cellular response is the rate of expression of a biochemical marker consisting of intracellular proteins of type p53, ATM, Chk1, Chk2, BRCA1, BRCA2, Nbs1, MRE11, Rad50, Rad51 and the histones. However, the radiation-induced expression cannot predict the functionality of said proteins (certain syndromes present a normal rate of expression even though the protein is mutated): these procedures are not functional tests.
Patent applications WO 01/90408, WO 2004/059004 and WO 2006/136686 (Commissariat à l'Energie Atomique) describe methods for observing DNA damage following ionizing irradiation. The first document discloses activities for incision of DNA lesions, but does not enable quantification of the enzymatic activities of DNA excision and resynthesis, nor of DSB repair. The two other documents describe quantitative evaluation of the capacity of a biological medium to repair DNA using super-coiled circular double-strand DNA (according to the third document: immobilized in a porous polyacrylamide hydrogel film). These methods do not directly concern DSB in situ in their physiological environment, and no correlation exists to validate their clinical application.
KR20030033519 proposes deducing sensitivity to radiation from the activity of a catalyst or of superoxide dismutase, KR20030033518 uses glutathione peroxidase or glucose 6-phosphate dehydrogenase. Such methods do not detect markers directly linked to DNA damage or repair.
Patent application US 2011/312514 (Dana Farber Cancer Institute) proposes using detection of FANCD2 foci as a marker. Patent application US 2007/0264648 (National Institute of Radiological Sciences) proposes to uses of DNA oligomers for predicting the appearance of side effects during radiotherapy. However, some radiosensitivities can be observed even though the concentration of FANCD2 foci is normal.
Patent applications US 2008/234946 and US 2012/041908 (University of South Florida et al.) describe a method for predicting radiosensitivity of cancerous cells, and not of healthy cells; moreover, it is based on genomic data and not on functional tests.
Patent application WO2014/154854 (Centre Hospitalier Universitaire de Montpellier) describes a method for predicting radiosensitivity of a subject through the use of at least one radiosensitivity biomarker. This method does not detect markers directly linked to DNA damage or repair; moreover, it is based on proteomic data. In addition, this patent application does not describe quantitative relations between the radiobiological data and the severity of tissular reactions.
Patent application WO 2013/187973 (University of California) describes systems and methods for determining the radiosensitivity of cells and/or of a subject with respect to a control population. More particularly, said method includes the radiation of a biological sample, the detection and quantification of radiation-induced foci within erythrocyte cells, lymphocytes or primary cells, resulting from a blood sample through the use of one or more detection markers selected from a set of markers including anti-pH2AX, anti-MRE11 and anti-ATM. Quantification of the radiation-induced foci at various post-irradiation observation times of less than two hours enables determination of the repair kinetics thereof, which is empirically correlated with the radiosensitivity of the subject. However, analysis of foci in lymphocyte cells is very difficult owing to their small nucleus. Moreover, said method does not therefore allow a practitioner to take decisions regarding patient treatment.
Patent U.S. Pat. No. 8,269,163 (New York University School of Medicine) describes a large number of proteins which can be used as markers in order to easily and rapidly appreciate the importance of accidental exposure to ionizing radiation to which a person has been subject, in order to sort patients and direct them towards an appropriate emergency treatment. Said patent relates to biological dosimetry (determination of accidental dose) while the detection of radiosensitivity is carried out using a known dose.
Patent application WO 2010/88650 (University of Texas) describes methods and compositions for identifying cancerous cells which are either sensitive or resistant to a particular radiotherapy treatment; it is therefore not applicable to all radiotherapy treatments.
Patent application WO 2010/136942 (Philips) describes a global method for monitoring a patient during radiotherapy, using biomarkers. The method comprises obtaining at least one descriptor derived from an image extracted from an imaging procedure, wherein the descriptor belongs to a tissue of interest for which radiotherapy is intended, or to a tissue in the vicinity of said target volume. The method further comprises the selection of at least one biological marker specific to a disease, suitable for detecting or quantifying side effects of radiotherapy in the tissue area of interest. The method further comprises the obtaining of at least one measurement value in vitro of the biomarker specific to the selected disease. The method further comprises processing the at least one descriptor of the at least one biomarker value in vitro by means of a correlation technique, resulting in an output signal indicative of the radiotoxicity in the tissue region of interest. However, the teaching of said patent only takes account of the dependent toxicity of the tissue and not that of the individual, and is mainly based on image analysis.
Patent application WO 2010/109357 describes a method and apparatus for scheduling of an adaptive radiotherapy protocol based on optimization of the probability of complication in normal tissues and the probability of tumor control according to markers specific to each patient. The values of markers associated with normal tissues comprise the in vitro test values, the mass spectrometry signatures of proteins, and the medical history data of the patient. The in vitro test values may be of cellular, proteomic and genetic origin, such as, but not limited to, various cell counts, HB, CRP, PSA, TNF-alpha, ferritin, transferrin, LDH, IL-6, hepcidin, creatinine, glucose, HbAlc, and the length of the telomeres. The markers from the patient history include earlier abdominal surgery, hormonal medications or anticoagulants, diabetes, age, and measurements related to tumor growth. Biomarkers not related to radiotoxicity are also envisaged, such as biomarkers associated with various forms of ablation or chemotherapy agents. However, individual radiosensitivity is not considered.
Despite this extensive prior art, the applicant notes that the patterns described above do not describe a method for quantification of individual radiosensitivity enabling an evaluation of the risk of post-radiotherapy tissular reactions, which could be employed for any patient and any type of ionizing radiation capable of inducing DSB, and which is predictive. Thus the problem of providing a method for predicting individual radiosensitivity still has no operational solution. The present invention aims to propose a novel method for predicting tissular and clinical radiosensitivity.